The present invention relates to an apparatus and method for the detection of test materials in small concentrations, especially the detection of pathogen indicators. In particular it relates to the detection of DNA, RNA and proteins in serum.
Historically, the diagnosis of diseases has depended upon clinical manifestations. However, new techniques of detecting diseases have been developed with the advent of nucleic acid and monoclonal antibody detection methods. The detection of nucleic acid has been used for diseases associated with abnormal gene products, such as anemia, Huntington""s disease and certain thalassemia mutations. In addition, the detection of nucleic acid has been used for bacterial and viral diseases, such as Human Immunodeficiency Virus (HIV). Moreover, monoclonal antibody detection methods have gained acceptance for the identification and differentiating of certain diseases such as cancers.
As appreciated by those skilled in the art, the detection of a pathogen indicator has applicability to the detection of certain diseases associated with abnormal genes, certain diseases associated with the presence of an identifiable nucleic acid sequence and certain diseases associated with the immune system. The pathogen indicator described herein includes DNA, RNA, antibody, antigen, and other proteins.
Known manual pathogen indicator detection methods in research and clinical laboratories tend to have low accuracy, low sensitivity and are subject to human error, both in carrying out the methods and in interpreting the results. Other methods, e.g. culturing methods, are not suitable for many diseases. For example, tuberculosis has a very slow growth rate, which makes detection not easy or even not possible.
U.S. Pat. No. 5,753,439 to Smith et. al. describes a method to detect characteristic dinucleotide and trinucleotide acid sequences, to determine target sequences and to screen for genetic defects and disorders associated with the sequences. The assays are conducted on solid surfaces allowing for multiple reactions to be conducted. However, the method does not provide a control process to provide assurances that the results are accurate and sensitive to determining if there is an error in the method.
U.S. Pat. No. 5,824,478 to Muller describes a method wherein a sample is contacted with a detector probe and a capture probe to form a detector probe-analyte-capture probe complex which is used to detect a target analyte in a sample. The target analytes, capture probes, and detector probes can be nucleic acids and polypeptides. However, the method does not provide a control process to provide assurances that the results are accurate and sensitive to determining if there is an error in the method.
In a standard enzyme ELISA method for immunoassay, a tray with a plurality of wells, e.g. 96 wells, containing appropriate antibodies is used. One method to eliminate error in this ELISA is to use a control. One of the wells can be used as a positive control (with a positive antigen), while the remaining wells can be used for testing patient""s sera. After addition of the serum samples, the wells are washed and a second antibody, which carries an enzyme, is added to the wells. After washing again, a substrate is added. The substrate and enzyme react, with a color reaction. The color yield from the reaction is associated with the presence of an antigen. The method is rife with possibilities for error. Human error can lead to some wells being washed twice or not at all, having reagents added twice or not at all, or wells being inadvertently contaminated with extraneous materials. For example, over washing tends to flush all the components and create a false negative result, while an incomplete wash will provide detection from non-binding materials and yield false positive results. The control well can give no assurance that the results from any other well is indicative of the presence or otherwise of the pathogen indicator under investigation. Additionally, color differences from well to well give additional uncertainties with respect to interpretation of the results.
Most of the previous tests are demanding of time, skill and concentration. So much so, that in many jurisdictions the number of tests that can be conducted by one technician is limited by regulation. This serves to raise the cost of testing, as it is so labour dependent.
For all the above reasons, and more, a new method, apparatus and a kit for detecting a pathogen indicator is desirable, which is accurate, reproducible, and is sensitive to determining if there is an error in the method.
The present invention provides a method for detecting the presence of a test material in a test sample. The method comprises the steps of: (a) introducing a test sample and a control material into a test column, wherein the column has at least two snares, one of said snares having a control capture material; at least one of said snares thereon having a target capture material specific to a corresponding test material in the test sample for which the detection is being sought, so that the control capture material will bind with the control material to form a bound control material; and the target capture material will bind with the corresponding test material to form a bound material; (b) washing the test column to remove any materials which have not been bound to the capture materials; and (c) detecting the presence of bound materials on each of the snares. The method can further comprise adding a label material for each of the bound materials to form labeled bound materials and then detecting the presence of the labeled bound materials.
In another embodiment, the present invention provides a method for detecting the presence of a DNA sequence in a test sample. The method comprises the steps of: (a) denaturing a test sample to form a single strand target DNA sequence for which detection is being sought; (b) introducing the test sample and a first control single strand DNA sequence into a test column which has at least two snares, one of said snares having a first control single strand capture DNA sequence; at least one of said snares thereon having a target single strand capture DNA sequence specific to the corresponding target DNA sequence in the test sample; and wherein the target single strand capture DNA sequence will bind with the corresponding target DNA sequence in the test sample to form a double strand DNA sequence, and the first control single strand capture DNA sequence will bind with the first control DNA sequence to form a double strand control DNA sequence; (c) adding a wash solution to the column to remove unbound DNA; (d) adding an enzyme to the column to destroy single strand DNA; (e) adding a denaturing solution to separate the formed double strand DNA sequences, then adding a wash solution to remove denatured non-capture single strand sequences, so that the single strand capture DNA sequences re-form on each snare; (f) adding DNA probes to provide detectable labels for single strand capture DNA sequences formed in step (e); (g) adding a wash solution to the column to remove unbound DNA probes; and (h) detecting any signals from each snare. In step (b), the first single strand control DNA sequence can be added into said test sample prior to introducing the sample into the test column, or can be added into the test column separately from the test sample. Moreover, the method can further include adding a substrate which reacts with the labels to give off detectable signals.
Additionally, the method further comprises introducing a second control single strand DNA sequence into the test column; wherein the test column has a control snare thereon having a second control single strand capture DNA sequence.
In a further embodiment, the snares have more than one single strand capture DNA sequences on one single snare, and the labels are different for different single strand capture DNA sequences on one single snare so that different DNA sequences can be detected on one single snare.
In yet another embodiment, the method for detecting the presence of a DNA sequence in a test sample comprises the steps of: (a) providing a positive control single strand DNA sequence; (b) denaturing a test sample to form a single strand target DNA sequence for which detection is being sought; (c) adding the test sample and the positive control DNA sequence to a test column, wherein the column has at least two snares, one of said snares having thereon a first control single strand capture DNA sequence for binding to a portion of the positive control DNA sequence; at least one of said snares thereon having a target single strand capture DNA sequence specific to the corresponding target DNA sequence in the test sample, so that the positive control DNA sequence binds with the first control single strand capture DNA sequence wherein the bound positive control DNA sequence has a double strand portion and a single strand portion; and the target DNA sequence present in the test sample binds with the target single strand capture DNA sequence wherein the bound target DNA sequence has a double strand portion and a single strand portion; (d) adding a wash solution to the column to remove unbound DNA; (e) adding DNA probes to provide detectable labels for attachment to the single strand portion of the bound positive control DNA sequence and the single strand portion of the bound target DNA sequence formed in step (c); (f) adding a wash solution to the column to remove unbound DNA probes; and (g) detecting any signals each snare. In addition, the method can further include adding a substrate which reacts with the labels to give off detectable signals.
The positive control single strand DNA sequence is prepared from a target DNA sequence for which detection is being sought, by a process selected from the group consisting of (1) inserting a control DNA fragment into the target DNA sequence for which detection is being sought at a predetermined scission point; and (2) removing a small fragment of DNA from the target DNA sequence at a predetermined scission point.
Further more, step (c) of the method can further include adding a negative control DNA sequence to the test column; wherein the test column also has a control snare having thereon a second control single strand capture DNA sequence for binding to the negative control DNA sequence, so that the negative control DNA sequence binds with the second control single strand capture sequence to form a bound negative control DNA sequence. The negative control single strand DNA sequence is different from the target DNA sequence and different from the positive control DNA sequence.
In a further aspect, the present invention provides a method for detecting the presence of a RNA sequence in a test sample. A method for detecting the presence of a RNA sequence in a test sample comprises the steps of: (a) providing a positive control single strand DNA sequence; (b) adding a test sample and the positive control DNA sequence to a test column wherein the column has at least two snares, one of said snares having thereon a first control single strand capture DNA sequence for binding to the positive control DNA sequence; at least one of said snares thereon having a target single strand capture DNA sequence specific to the corresponding target RNA sequence in the test sample, so that the positive control DNA sequence binds with the first control capture DNA sequence to form a double strand positive control DNA sequence, and the RNA sequence present in the test sample binds with the target capture DNA sequence to form a double strand DNA/RNA complex; (c) adding a wash solution to the column to remove unbound positive control DNA and target RNA; (d) adding an enzyme to the column to destroy single strand DNA and RNA; (e) adding a denaturing solution to separate the formed double strand control DNA sequence and double strand DNA/RNA complex, then adding a wash solution to remove denatured non-capture single strand DNA and RNA sequences, so that the single strand capture DNA sequences re-form on each snare; (f) adding DNA probes to provide detectable labels for single strand capture DNA sequences formed in step (e); (g) adding a wash solution to the column to remove unbound DNA probe; and (h) detecting any signals from each snare. In addition, the method can further include adding a substrate which reacts with the labels to give off detectable signals.
In one embodiment, the positive control single strand DNA sequence is different from the target RNA sequence and the first control single strand capture DNA sequence is different from the target single strand capture DNA sequence. In addition, the DNA probes used in step (f) are different for the first control capture and the target capture sequences.
In another embodiment, the positive control single strand DNA sequence has a portion which has the same sequence to a portion of the target RNA sequence. The first control single strand capture DNA and the target single strand capture DNA have a common sequence at a portion of the capture sequences, so that a common DNA probe is used in step (f) for detection of the re-formed control and target capture sequences.
In a further embodiment, step (a) of the RNA detection method further includes providing a negative control single strand DNA sequence which is different from the target RNA sequence and different from the positive control DNA sequence. Step (b) further includes adding the negative control DNA to the test column which also has a control snare having thereon a second control single strand capture DNA sequence. The second control capture DNA sequence partially matches the negative control DNA sequence so that the negative control DNA sequence binds with the second control capture DNA sequence to form a double strand DNA sequence which also has unbound single strand portions. Step (f) DNA probes do not match re-formed partial second control single strand capture DNA sequence formed in step (e), and no binding occurs between them. Therefore, in step (h) no signal is detected from the second control snare under normal conditions.
In yet another embodiment, the second control single strand capture DNA sequence is different from the target capture DNA and the first control capture DNA sequences.
In an additional embodiment, the first control capture DNA, the second control capture DNA and the target capture DNA have a common sequence at a portion of the capture sequences. A common DNA probe is used in step (f) for detection of the re-formed control and target capture sequences.
In another aspect, the present invention provides a column for analysis of a test material, wherein the column has at least two snares, one of said snares having thereon a first control capture material for detecting the presence of a first control material, and at least one of said snares having thereon a test capture material for detecting a test material for which detection is being sought. The column can also comprise at least two chambers, each chamber having a snare, one of said chambers having a first control capture material on the snare for detecting the presence of a first control material, and at least one of said chambers having a test capture material on the snare for detecting the test material for which detection is being sought.
In a further embodiment, the chambers have a connecting means to connect different chambers in order, and the chambers are connected along the longitudinal axis of the chamber through the connecting means. Alternatively, the chambers can be placed side-by-side. Furthermore, the snares can reside on a snare tray which is in a plane transverse to a longitudinal stem. When the stem rotates, the snares will be conveyed to the sample station, reagent stations, and detection stations.
Additionally, the column further has a chamber or a snare having a second control material on the snare for detecting the presence of a second control material.
In a further aspect, the present invention provides a kit. The kit comprises (a) a column for analysis of a test material, wherein the column has at least two snares, one of said snares having thereon a first control capture material for detecting the presence of a first control material, and at least one of said snares having thereon a capture material for detecting a test material for which detection is being sought; (b) reagents for detecting the presence of the test materials.
In an additional aspect, the present invention provides an apparatus for detection of a test material in a sample. The apparatus comprises (a) a column handler system which comprises column holders and an column off-loader which unloads a test column; (b) a sample station for adding a sample to a test column; (c) a reagent station; (d) a detection station for detection of a control material and the test material in the testing column; and (e) a carousel comprising a rotating frame which carries the column holder; wherein the carousel conveys the test column to the sample station, reagent station, and detection station.
In another embodiment, the detection station has a plurality of detectors for detecting the presence of one or more control materials and one or more test materials on the snares.
In an additional aspect, the present invention provides a sample dispenser comprising: (a) a sample holder which comprises a side wall, and a bottom connected to the side wall; wherein said bottom has a bore which is sealed with a film capable of being punctured; and (b) a puncturer thereon having a delivering spout for puncturing said film of the bore.
The puncturer of the sample dispenser connects to a test column. When the sample holder is situated on top of the puncturer and lowered toward the column, the puncturer punctures the film of the sample holder to dispense a liquid sample in the sample holder into the test column.